Tandem Affinity Purification and Identification of Heterotrimeric G Protein-Associated Proteins

Abstract

Heterotrimeric G proteins are the main signal-transducing molecules activated by G protein-coupled receptors. Their GTP-dependent activation leads to the regulation of different effectors such as adenylyl cyclases, phospholipases, and RhoGEFs. To understand the full biological consequences of GPCR signalling and to further understand the cross-talk with other signalling pathways, the complement of proteins associating with heterotrimeric G proteins needs to be identified. Here we describe our mass spectrometry-based proteomic approaches for the study of Gβγ and Gα protein complexes. This approach is predicated on the establishment of mammalian cell lines constitutively or inducibly expressing affinity-tagged versions of Gβγ or wild-type and constitutively active Gα subunits, respectively.